鄂西特有植物提取物的杀虫活性

首次报道悬铃木叶苎麻、龟背竹、李叶绣线菊、扇脉杓兰、鞘柄木、青牛胆、仙茅和大叶金腰的杀虫活性。质量分数为1%的(下同)千里光茎、李叶绣线菊花、龟背竹、扇脉杓兰茎叶、悬铃木叶苎麻和鞘柄木叶甲醇提取物对舍蝇Musca domestica vicinaMacquart 48 h的致死率在70%以上,其中千里光茎为96.55%。仙茅、扇脉杓兰和李叶绣线菊花对赤拟谷盗Tribolium castaneum(Herbst)种群60 d繁殖抑制率达50%以上,其中最高仙茅为83.52%。仙茅和李叶绣线菊花对赤拟谷盗30 d的毒杀率在60%以上。悬铃木叶苎麻对斜纹夜蛾Spodoptera lituraFabricius 3龄幼虫48 h的拒食率为84.00%。李叶绣线菊花、青牛胆、悬铃木叶苎麻和大叶金腰对亚洲玉米螟Ostrinia furnacalis(Guen?e)3龄幼虫具较好拒食效果,其中,李叶绣线菊花48 h对亚洲玉米螟3龄幼虫的拒食率为81.17%,这几种植物还引起试虫体重不增加、化蛹延迟、畸形蛹或部分蛹死亡现象。所筛选植物对致倦库蚊Culex pipiens quinguefasciatusSay 4龄幼虫48h的毒杀率均低于20%。以甲醇冷浸提取法对所采集的植物材料进行冷浸提取时,提取率分布在1.92%至12.92%之间。     

Optimal methods for evaluating antimicrobial activities from plant extracts

The search for antimicrobial agents from plants has been a growing interest in the last few decades. However, results generated from many of these studies cannot be directly compared due to the absence of standardization in particular antimicrobial methods employed. The need for established methods with consistent results for the evaluation of antimicrobial activities from plant extracts has been proposed by many researchers. Nevertheless, there are still many studies reported in the literature describing different methodologies. The aim of this study was to find optimal methods to give consistent quantitative antimicrobial results for studying plant extracts. Three different agar-based assays (pour plate disc diffusion (PPDD), streak plate disc diffusion (SPDD) and well-in agar (WA)) and one broth-based (turbidometric (TB)) assay were used in this study. Extracts from two plant species (Duabanga grandiflora and Acalypha wilkesiana) were tested on two bacterial species, namely Escherichia coli and Staphylococcus aureus. Amongst the agar-based assays, PPDD produced the most reproducible results. TB was able to show the inhibitory effects of the test samples on the growth kinetic of the bacteria including plant extracts with low polarity. We propose that both agar- (i.e PPDD) and broth-based assays should be employed when assessing the antimicrobial activity of plant crude extracts.     

Polysaccharide microarrays for high-throughput screening of transglycosylase activities in plant extracts

Polysaccharide transglycosylases catalyze disproportionation of polysaccharide molecules by cleaving glycosidic linkages in polysaccharide chains and transferring their cleaved portions to hydroxyl groups at the non-reducing ends of other polysaccharide or oligosaccharide molecules. In plant cell walls, transglycosylases have a potential to catalyze both cross-linking of polysaccharide molecules and grafting of newly arriving polysaccharide molecules into the cell wall structure during cell growth. Here we describe a polysaccharide microarray in form of a glycochip permitting simultaneous high-throughput monitoring of multiple transglycosylase activities in plant extracts. The glycochip, containing donor polysaccharides printed onto nitrocellulose-coated glass slides, was incubated with crude plant extracts, along with a series of fluorophore-labelled acceptor oligosaccharides. After removing unused labelled oligosaccharides by washing, fluorescence retained on the glycochip as a result of transglycosylase reaction was detected with a standard microarray scanner. The glycochip assay was used to detect transglycosylase activities in crude extracts from nasturtium (Tropaeolum majus) and mouse-ear cress (Arabidopsis thaliana). A number of previously unknown saccharide donor-acceptor pairs active in transglycosylation reactions that lead to the formation of homo- and hetero-glycosidic conjugates, were detected. Our data provide experimental support for the existence of diverse transglycosylase activities in crude plant extracts.     

Fluorometric microassays for the determination of cathepsin L and cathepsin S activities in tissue extracts.

We established a continuous semi-microassay, and for large-scale studies both a stopped and a continuous microtiter plate assay for the fluorometric determination of cathepsin L and cathepsin S activities in body fluids, tissues or cell extracts in the presence of cathepsin B. For the detection of enzymatic activities we used the synthetic substrate Z-Phe-Arg-AMC, and for discrimination between cathepsin L, S and cathepsin B the specific inhibitor CA-074 for blocking interfering cathepsin B activities was applied. Furthermore, we took advantage of the stability of cathepsin S at pH 7.5 for further differentiation between cathepsin L and cathepsin S activities. The kinetic assays were characterized in terms of imprecision, analytical sensitivity, accuracy and substrate concentration. The within-run coefficients of variation were found to be 4.9%-7.2% for the continuous semi-microassay, 10.3%-11.7% for the stopped, and 4.5%-11.8% for the continuous microtiter plate assay. The between-days coefficients of variation for the continuous semi-microassay were 8.1%-8.9%, while for the stopped and continuous microtiter plate assays the coefficients were 11.2%-13.5% and 5.8%-12.2%, respectively. Compared to the continuous semi-microassay, the stopped and the continuous microtiter plate assays showed 3-fold and 11-fold higher sensitivity, respectively. Comparison between the continuous enzyme activity assays at substrate concentrations of 40 microM and 200 microM demonstrated a significant correlation of r = 0.97 and r = 0.99, respectively. The newly developed microtiter plate assay will allow efficient, sensitive and high precision determination of cathepsin L and cathepsin S activities in large-scale studies of cysteine-cathepsin dependent diseases.     

A rapid method for determination of nitrate in soil and plant extracts

A rapid colourimetric method of determination of nitrate was modified. Proposed modifications eliminated the use of barium sulphate and introduced diazotization of sulphanilamide by the nitrite ion obtained by the reduction of nitrate and subsequent coupling with N-1-naphthyethelenediamine dihydrochloride. Introduction of filtration in place of centrifugation of coloured solution simplified the procedure. Determinations were highly reproducible with coefficient of variation of 2.2 and 2.9% for soil and plant extracts respectively.     

Cytotoxic and antiviral activities of Colombian medicinal plant extracts of the Euphorbia genus

Forty-seven plant extracts of 10 species of the genus Euphorbia (Euphorbiaceae) used by Colombian traditional healers for the treatment of ulcers, cancers, tumors, warts, and other diseases, were tested in vitro for their potential antitumour (antiproliferative and cytotoxic) and antiherpetic activity. To evaluate the capacity of the extracts to inhibit the lytic activity of herpes simplex virus type 2 (HSV-2) and the reduction of viability of infected or uninfected cell cultures, the end-point titration technique (EPTT) and the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric assay were used, respectively. The therapeutic index of the positive extracts for the antiviral activity was determined by calculating the ratio CC50 (50% cytotoxic concentration) over IC50 (50% inhibitory concentration of the viral effect). Five of the 47 extracts (11%) representing 3 out of 10 Euphorbia species (30%) exhibited antiherpetic action; the highest activity was found in the leaf/stem water-methanol extracts from E. cotinifolia and E. tirucalli. The therapeutic indexes of these two plant species were > 7.1; these extracts exhibited no cytotoxicity. Six extracts (13%) representing 4 plant species (40%) showed cytotoxic activity. The highest cytotoxicity was found in the dichloromethane extract obtained from E. cotinifolia leaves and the CC50 values for the most susceptible cell lines, HEp-2 and CHO, were 35.1 and 18.1 microgram/ml, respectively.     

Simple kinetic determination of trace amounts of ascorbic acid in plant extracts

Aa kinetic method of ascorbic acid assay based on the lag produced during the evolution of horse radish peroxidase-catalyzed oxidation of guaiacol or of homovanillic acid by hydrogen peroxide, is described and applied to ascorbic acid determination in Sedum album extracts.     

Pest-managing activities of plant extracts and anthraquinones from Cassia nigricans from Burkina Faso

Insecticidal activity of eight plants collected from Burkina Faso was studied using mosquito (Ochlerotatus triseriatus), Helicoverpa zea and Heliothis virescens larvae and adult white fly (Bemisia tabaci). The n-hexane, ethyl acetate and methanol extracts of Pseudocedrela kotschyi, Strophantus hispidus, Securidaca longepedunculata, Sapium grahamii, Swartzia madagascariensis, Cassia nigricans, Jatropha curcas and Datura innoxia were used in this study. Extracts were tested at 250 microg/mL concentration. All three extracts of C. nigricans, J. curcas (skin and seeds) and D. innoxia exhibited 100% mortality on fourth instar mosquito (O. triseriatus) larvae. In addition, the n-hexane and ethyl acetate extracts of S. hispidus, S. longepedunculata, S. grahamii showed 100% mortality. The ethyl acetate extract of S. madagascariensis was the most active on adult white fly and exhibited 80% mortality. Extracts of all other plants exhibited 30-50% mortality on B. tabaci. In the antifeedant assays against H. zea and H. virescens, the MeOH extracts of C. nigricans, S. madagascarensis and S. hispidus were more effective against H. zea as indicated by 74% larval weight reduction as compared to the control. Since C. nigricans is commonly used in West Africa to protect grain storage from insects, we have characterized the insecticidal components present in its extract. Bioassay directed isolation of C. nigricans leaf extract yielded anthraquinones emodin, citreorosein, and emodic acid and a flavonoid, luteolin. Emodin, the most abundant and active anthraquinone in C. nigricans showed approximately 85% mortality on mosquito larvae Anopheles gambiaea and adult B. tabaci at 50 and 25 microg/mL, respectively, in 24 h. These results suggest that the extract of C. nigricans has the potential to be used as an organic approach to manage some of the agricultural pests.     

Radioimmunoassays for the differential and direct analysis of free and conjugated abscisic acid in plant extracts

Two radioimmunoassays have been developed which allow the parallel quantitation of free as well as conjugated natural (+)-abscisic acid (ABA) directly and separately, in unpurified plant extracts. The differential specificity of antisera has been achieved by coupling ABA through C₁ (for total ABA determination) or C₄ (for free ABA determination), respectively, to proteins to obtain the immunogenic conjugates. Compounds structurally related to ABA, such as, dihydrophaseic acid or phaseic acid, do not interfere with either of the assays, even when present in more than ten-fold excess. Other related compounds, such as, violaxanthin or xanthoxin, do not cross react at all. Both antisera respond to (+)-ABA but show very low immunoreactivity with (-)-ABA. As little as 27 pg of ABA (serum for free ABA) or 47 pg (serum for total ABA) may be detected and the measuring ranges are from 0.2–8 and 0.2–30 pmol, respectively. Average recoveries are greater than 99%. Using these assays, more than 100 samples can be assayed for free and conjugated ABA per day. Levels of free ABA, as determined by radioimmunoassay (RIA), correlated well with those reported in the literature. Levels of conjugated ABA were found to be generally higher than previously reported for ABA after alkaline hydrolysis of the extracts. Conjugated ABA accumulates during aging of leaves and levels of conjugated ABA up to 17-fold higher than those of free ABA have been detected in senescent leaves of Hyoscyamus niger L. Evidence was obtained for the presence of ABA conjugates other than the glucose ester in some plants.Abbreviations ABA abscisic acid - BHT 2,6-di-t-4-methyl phenol - BSA bovine serum albumin - HSA human serum albumin - RIA radioimmunoassay - TLC thin-layer chromatography - EDC 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide · HCl Part 11 in the series: Use of Immunoassay in Plant Science     

测定植物胞内游离钠离子的研究进展

植物耐盐机制的研究一直是植物抗性研究的焦点。近年来,随着生物学不断发展和新荧光标记技术的运用,胞内钠离子测定逐渐应用于植物盐胁迫研究中。该文论述了以下三方面问题:(1)分别介绍了SBFI、Sodium Green和Coro Na Green三种钠离子荧光指示剂:SBFI是一种双激发波长指示剂,其激发波长是340 nm/380 nm,发射波长是500 nm;Sodium Green和Coro Na Green是单波长指示剂,其激发波长分别是507 nm和492nm,发射波长分别是532 nm和516 nm;(2)比较了酯导入、酸导入、电穿孔和显微注射等几种常见荧光指示剂载入胞内方法的优缺点,重点介绍了一种无损伤低温抑制酯酶法:先将荧光指示剂在缓冲液中低温(4℃)处理2 h,随后回到常温(20℃)在不含荧光指示剂的缓冲液中孵育2 h;(3)阐述了胞内离子浓度计算公式,包括单波长测定公式、双波长比率测定公式。     

Pharmacological activities of Vitex agnus-castus extracts in vitro.

The pharmacological effects of ethanolic Vitex agnus-castus fruit-extracts (especially Ze 440) and various extract fractions of different polarities were evaluated both by radioligand binding studies and by superfusion experiments. A relative potent binding inhibition was observed for dopamine D2 and opioid (micro and kappa subtype) receptors with IC50 values of the native extract between 20 and 70 mg/mL. Binding, neither to the histamine H1, benzodiazepine and OFQ receptor, nor to the binding-site of the serotonin (5-HT) transporter, was significantly inhibited. The lipophilic fractions contained the diterpenes rotun-difuran and 6beta,7beta-diacetoxy-13-hydroxy-labda-8,14-dien . They exhibited inhibitory actions on dopamine D2 receptor binding. While binding inhibition to mu and kappa opioid receptors was most pronounced in lipophilic fractions, binding to delta opioid receptors was inhibited mainly by a aqueous fraction. Standardised Ze 440 extracts of different batches were of constant pharmacological quality according to their potential to inhibit the binding to D2 receptors. In superfusion experiments, the aqueous fraction of a methanolic extract inhibited the release of acetylcholine in a concentration-dependent manner. In addition, the potent D2 receptor antagonist spiperone antagonised the effect of the extract suggesting a dopaminergic action mediated by D2 receptor activation. Our results indicate a dopaminergic effect of Vitex agnus-castus extracts and suggest additional pharmacological actions via opioid receptors.     

Effect of salinity on plant growth and biological activities of Carthamus tinctorius L. extracts at two flowering stages

In the present study, we were interested in the effect of salt stress on phenolic and carotenoid contents, antioxidant and antimicrobial activity of two varieties of Carthamus tinctorius (Jawhara and 104) flowers. For this purpose, C. tinctorius flowers from plants grown under four saline treatments (0, 5, 10 and 15 g/L NaCl) were collected at two development stages. As salinity increased up to 10 g/L, results showed that total phenols, flavonoids, condensed tannins and carotenoid contents increased with salinity. Such variability might be of great importance in terms of valorizing this plant as a source of naturally secondary metabolites. Furthermore, our results showed an enhancement of antioxidant activity which was evaluated by four different test systems (DPPH, β-carotene–linoleic acid, chelating and reducing power assays) with increasing stress severity. Obtained results showed that, for the two varieties, salt effect was more pronounced at post flowering stage than full flowering one. The sensitivity test of the methanolic extracts of the harvested flowers was applied against seven human pathogenic bacteria and three yeast strains. Salinity reduced significantly the antimicrobial activity of flower extracts.